This application relates to the field of sample processing systems and methods of processing biological samples. The present invention may be directed to the automated processing, treatment, or even staining of samples arranged on carriers, such as microscope slides or similar plane, rectangular sample carriers, and in some embodiments, directed to the continuous or batch processing of samples and carriers, as well as washing elements of a sampling system. Embodiments may further relate to control systems for sample processing and data acquisition, data maintenance, and data retrieval for sample processing. Applications to which the present invention may especially relate include immunohistochemistry, in-situ hybridization, fluorescent in-situ hybridization, special staining, and microarrays, as well as potentially other chemical and biological applications.
In this application, the term “staining” is used for the end product of the process, by which certain parts of the sample may be stained, i.e. have obtain a different color, either in the optic range or in another electromagnetic range, such as ultra violet, or the staining may be a detectable, preferably automatically detectable, change in properties, such as fluorescent properties, magnetic properties, electrical properties or radioactive properties. To obtain the staining, the sample normally must undergo a series of treatment steps, such as—but not limited to—washing, binding of reagents to the specific parts of the sample, activation of the reagents, etc. and each treatment step may include a plurality of individual treatments.
Sample processing in immunohistochemical (IHC) applications and in other chemical and biological analyses may require one or a number of various processing sequences or protocols as part of an analysis of one or more samples. The sample processing sequences or protocols may be defined by the individual or organization requesting an analysis, such as a pathologist or histologist of a hospital, and may be further defined by the dictates of a particular analysis to be performed.
In preparation for sample analysis, a biological sample may be acquired by known sample acquisition techniques and may comprise, for example, in IHC applications, tissues generally or, even, in some applications one or a plurality of isolated cells, such as in microarray samples, and may be presented on a microscope slide or a similar plane, rectangular sample carrier. Furthermore, the sample may be presented on the slide or other carrier variously and potentially in some form of preservation. As one example, a sample such as a layer or slice of skin may be preserved in formaldehyde and presented on a slide with one or more paraffin or other chemical layers overlying the sample.
Immunologic applications, for example, may require processing sequences or protocols that comprise steps such as de-paraffinization, target retrieval, and staining, especially for in-situ hybridization (ISH) techniques. Previously, in some applications, these steps may have been performed manually, potentially creating a time-intensive protocol and necessitating personnel to be actively involved in the sample processing. Attempts have been made to automate sample processing to address the need for expedient sample processing and a less manually burdensome operation. However, such previous efforts may have not fully addressed the needs for an automated sample processing system. Such prior efforts to automate sample processing may be deficient in several aspects that prevent more robust automated sample processing, such as: the lack of sufficient computer control and monitoring of sample processing; the lack of information sharing for processing protocol and processing status, especially for individual samples; the lack of diagnostic capabilities; and the lack of real-time or adaptive capabilities for multiple sample batch processing.
The staining procedure is laborious and uses many different reagents. The staining protocol may include the following steps: de-paraffinization, washing, antigen retrieval, endogenous biotin or enzyme blocking, incubation with immunological reagents, molecular probes, secondary visualization reagents and various chromogen reagents, washing steps and counterstaining.
The use of fixed, paraffin-embedded tissue sections for immunohistochemistry staining permits localization of a wide variety of antigens while retaining excellent morphologic details. However, most chemical fixatives produce denaturation or masking of many antigens. Specifically, the denaturing effects of tissue fixation often render target epitopes on antigens unavailable for binding by antibodies during IHC.
Antigen retrieval is a widely accepted method for heat-assisted retrieval of antigens in fixed paraffin-embedded tissues prior to IHC staining. The introduction of antigen retrieval by heating tissue sections in a microwave oven, pressure cooker, or other heating devices before immunostaining has been an important breakthrough in improving results of staining by IHC. Although, many antigens can be retrieved when heating is performed in distilled water, there are several antigens that require use of buffers during the heating process. However, thus far, no single chemical has been identified that makes antigen retrieval effective for all antigens.
Many studies have shown an inverse relationship between the temperature and the duration of time that a sample is heated, such that low temperatures need prolonged incubation times for antigen retrieval compared to high temperatures that need shorter incubation times for antigen retrieval.
Generally, higher temperatures of about 100° C. can be achieved with water-based aqueous solutions. However, the duration of time needed for antigen retrieval is about 20-60 minutes and does not work for all antigens, resulting in poor staining for some antigens. Pressurized devices are often more effective for antigen retrieval as they enable the temperatures to be even higher, i.e., about 120° C. Most pressurized devices, however, generally require about 40-50 minutes for effective antigen retrieval. At least a portion of this time is used for the cooling process after the antigen retrieval heating step has been completed. Because the heating step is performed under pressure, the contents of the pressure chamber must be allowed to cool sufficiently so that the internal pressure of the chamber decreases prior to opening the chamber, as this can result in explosive boiling of the liquid inside and would pose a significant risk to the user. Typically the pressure chamber is allowed to passively cool under ambient conditions for at least 25-30 minutes before the container can be opened. This long duration may pose a problem for certain users that require faster processing times. Furthermore, not every laboratory is willing to invest in the time and money necessary to set up a high pressure method of antigen retrieval. Because the water bath method of antigen retrieval is easier to perform, many laboratories still rely on this method, although results are frequently inferior to the pressure method. Also there are some antigens that are more sensitive to higher temperatures and become denatured when exposed to 120° C. for prolonged periods of time. These antigens could benefit from lower temperatures or shorter incubation times to prevent denaturation.
Therefore, there is a need for improved, easy to use, and cost effective methods of antigen retrieval that facilitate effective antigen retrieval in a shorter duration of time, while at the same time, avoid problems associated with current antigen retrieval methods, such as denaturation of antigens and poor staining during IHC for disease detection.
In one embodiment, this invention provides compositions, methods and devices for heat-induced antigen retrieval where effective antigen retrieval can be achieved in a short duration of time without the need for a pressurized system.
The inventors have discovered that the addition of certain chemicals to antigen retrieval solutions may enable effective antigen retrieval in a shorter duration of time relative to antigen retrieval in absence of such chemicals, without the need of a pressurized system.
Viscosity generally refers to the measurement of a fluid's internal resistance to flow. Viscosity of a liquid, as used herein, can be measured by one or more methods known in the art and can be designated in units of centipoise or poise or any other acceptable measurements. Viscosity can be easily measured using one of many viscosimeters known in the art. Examples of viscosimeters include, but are not limited to, the Saybolt universal and the Saybolt Furol viscosimeters, which measure viscosity of liquids by allowing a measured volume of a liquid to flow through an orifice of specified dimensions and measuring the time in seconds that it took to get through the orifice. The Irany, Zahn and Redwood viscosimeters operate on the same principal. Other examples of viscosimeters include the Brookfield viscosimeter which includes a disc which is rotated in the liquid to be tested and the drag is noted and read directly in centipoise. Viscosities found herein were obtained from published sources, for example Handbook of Chemistry and Physics or The Merck Index.
Methods for mixing reagents and liquids are well known—but several aspects are important in the context of IHC and ISH instruments wherein the mixing in some processes can be cumbersome. By providing a sample processing apparatus having an automated mixer integrated therein, these types of staining processes can be performed automatically instead of requiring human interaction or manual performance of some process steps, thereby achieving a much more automated process, and the quality of the staining process may be improved as a desired degree of mixing of reagents may be provided as well as an optimal application time window for a deteriorating mixture may be reached.
An on-board mixing device for an automated biological sample processing apparatus should be able to mix multiple reagents and mixtures. Non-limiting examples include: dilution of chromogens concentrates, mixing and dilution of two, three or four component enzyme chromogen reagents, dilution of buffer concentrates, dilution of immunological reagents with dilution buffer, dilution of visualization reagents with dilution buffers, mixing several visualization reagents with dilution buffer or dilution of enzyme blocking reagents, dilution of biotin blocking reagents or mixing and dilution of counterstaining reagents.
Unfortunately, during the mixing, several problems may arise. Some problems arise due to the complex use of reagents in the staining procedure. Below a few non-limiting examples are listed in more detail:                1. The chromogen reagents (e.g. DAB, AEC, fast red etc) often comes as concentrated reagents in organic or high viscosity solutions and needs to be diluted prior to being applied to the sample. Chromogens like the Fast Red alkaline phosphatase chromogen are made ready for use by mixing and dilution of two or three reagents, which are very different in nature with regard to salt content, viscosity and density. Furthermore, the resulting mixtures are unstable over time and need to be used within a short time. Some chromogens suitable for e.g. horseradish peroxidase, like DAB and AEC, are easily oxidized when exposed to air during e.g. vigorous mixing or dilution.        2. The enzyme chromogens and counter-stain reagents like e.g. hematoxylin, are semi oxidized and can contain precipitates and solids. By further oxidation or slight change in pH, the reagents can further precipitate.        3. Antibody and enzyme containing reagents often contain stabilizing proteins and or detergents, which causes the solution to foam when being shaken or stirred. Many proteins cannot easily tolerate to be exposed to the hydrophobic air in foam. Wash buffers can contain detergents, which can foam when shaken or stirred. The foam can spread to other compartments of the instrument in an unwanted and unpredictable way. Mixing of some reagents like e.g. the HRP chromogens and peroxide reagents can result in the formation of small bubbles. These can generate foam or bubbling on the surface of the mixture.        4. Spill over/carry over must be avoided. The staining process is characterized by using many, complex and very different reagents and buffers and in many different dilution ratios and mixtures. Some of the reagents or buffers are incompatible with each other. In the event of cross contamination due to e.g. carry over, the reagents may be ruined within seconds or solids can precipitate, making the staining unsuccessful. For example, enzyme containing reagents can not be mixed with the corresponding chromogens, or high salt concentrates may not be mixed with e.g. proteins containing mixtures, or organic solvents can not be mixed with protein containing mixtures, or highly pH buffered wash buffers can not be mixed with low buffered mixtures without significantly altering the properties of the reagents. Accordingly the cleansing and washing of the mixing device need to be very efficient.        
As the procedures are very complex, and the instrument uses many different protocols, one cannot predict the result of reagent carry-over or unplanned mixing of reagents. Consequently, a mixing device for an automatic biological sample processing apparatus should ideally be very efficient and be designed for a variety of reagent mixing protocols and sequences.
During staining, build-up of small fouling layers on the various surfaces will rapidly cause problems, as the typical staining protocol calls for many mixing and dilution steps. Consequently, the mixing device should have a minimum of surface area and very smooth surfaces. Furthermore, the mixing device should ideally be able to mix very different volumes of reagents in both small and large volumes ratios. In other words, the degree of dilution and mixing ratios of reagents may vary from small to high ratios. In summary, the mixing device should ideally allow: mixing of small and large volumes; mixing reagents with different viscosities and densities; mixing of immiscible or nearly immiscible reagents; no fouling of mixing rods or similar due to precipitated material; easy escape of formed gasses during mixing; prevention of foaming of e.g. detergent or protein containing reagents; low build-up of debris or fouling on the device surfaces; easy emptying and washing—regardless of volumes; and very low reagent carry-over. No present mixing system for automated biological sample processing apparatus truly fulfills the above-mentioned properties. On-the-slide mixing does not allow for very large ratios of dilution. Neither does it allow for efficient mixing of reagents with very different densities or viscosities.
Further, a defined staining protocol may include one or more defined temperatures. Important, therefore, for many IHC applications, and many sample processing sequences and protocols, generally, are temperature characteristics associated with the sample, sample carrier, and the processing environment. Traditional sample processing technology has provided temperature control through heating devices that heat an entire set of sample carriers in the sampling processing system. Other technologies, such as the sample processing system described in U.S. Pat. No. 6,183,693, may provide heating devices for individual sample carriers that are individually controlled to heat the slides. However, each of these traditional sample processing systems may lack a desired degree of temperature control or temperature tolerances.
Inadequacies in temperature control of traditional technologies may include uncontrolled cooling. Traditional systems may only provide ambient cooling when the heating devices are off. Ambient cooling is not considered active control and may not meet protocol temperature requirements or may not otherwise be optimal. Although heating and heat control may be features of such systems, controlled cooling of the samples, sample carriers, and processing environments may not always be adequately addressed. Cooling techniques such as hooded fans may be incorporated in some traditional technologies. However, these devices can lack sufficient capabilities of temperature control to meet certain protocol requirements, especially temperature tolerances for samples, sample carriers, reagents, and ambient system temperature.
Traditional systems may even lack temperature control, perhaps as related to temperature tolerances generally, as such tolerances may not be adequately maintained during ambient or other traditional cooling, or during processing sequences or events, generally. In some protocols, for example, the temperature tolerances during non-heating periods may be such that uncontrolled temperature changes may produce undesirable results during the processing sequence. Other IHC processes of the protocol may be adversely affected by uncontrolled temperature changes, the degree of temperature change, and temperature changes outside of preferred tolerances. The lack of temperature control may actually dissuade technologists from employing automated processing sequences or protocols, especially IHC sequences, that may be dependent upon a particular temperature tolerance and the amount of temperature change during a processing sequence.
Certain types of temperature control may not have even been addressed in traditional sample processing system technologies. Reagents can play a vital role in the staining sequence of many processing protocols. The quality of the reagents, therefore, may be important for adequate sample processing. Reagents, for example, can have a certain shelf life that may be limited if maintained at undesirable temperatures such as the typical ambient temperatures of traditional biological sample processing systems and the laboratories housing such systems. Traditional technologies may lack the temperature control needed to optimally preserve the reagents stored in the processing system that are often subject to inadequate or changing ambient temperatures of such systems and the laboratory environment.
Sample processing apparatuses for staining and treating samples by means of probes normally comprises a first section or station for containing one or more reagent containers, such as bottles or vials; a second section or station for mounting slides, a probe arranged to aspirate a portion of reagent from a selected reagent container and dispensing the reagent to a slide on which the sample is arranged and a drive means for moving the probe between the various sections.
Past efforts at automated sample processing for samples presented on carriers such as slides, such as U.S. Pat. No. 6,352,861 and U.S. Pat. No. 5,839,091, have not afforded the various advantages and other combinations of features as presented herein. U.S. Pat. No. 5,948,359 discloses an apparatus of the above mentioned type, wherein the first station comprises a vial holder for holding 40 or more vials in order to provide a wide range of different reagents adapted for different staining purposes, and thereby the possibility of automatically staining a large number of slides requiring different staining processes. In practice it is very important that the apparatus facilitates that many different staining processes can be performed at the same time in the apparatus, because this avoids the necessity of batching samples requiring the same procedure or other treatment with reagents, and processing each batch individually.
Even though automated biological staining apparatuses are known in the prior-art, these conventional apparatuses do not provide for sample pre-treatment. Biological samples, such as tissue samples, must be prepared before the staining can be performed. The tissue slides are subjected to a pre-treatment process depending upon the type of staining process that is to be performed on the tissue. This pre-treatment could include de-paraffinization or target retrieval. The preparation of the tissues on the slides is often carried out manually in the laboratory before they are loaded into the automatic staining instrument. This pre-treatment includes immersing the slide in a buffer or other types of processing liquid for a predetermined amount of time and temperature. Unfortunately, however, this manual preparation is cumbersome and the pre-treatment may be insufficient, since it is critical that the amount of time and the temperature of the liquid must be observed very precisely in order to achieve the correct pre-treatment result.
In the U.S. Pat. No. 5,839,091, an automated staining apparatus is disclosed wherein a plurality of biological samples accommodated on microscope slides may be processed. However this instrument does not provide a processing tank for pre-treatment of the slides.
Some staining processes involve the use of hazardous materials, such as toxic materials. These materials may be collected in special containers in order to ensure safe handling of the waste material. However, this does not sufficiently protect the laboratory environment in which the apparatus is placed from being contaminated with toxic material. Moreover, in some staining processes or other treatments in the apparatus, heat is applied. This increases the risk of vaporizing reagents which then may escape to the outside of the apparatus.
In the apparatuses known in the art, a protective hood or similar plastic cover is put over the staining apparatus in order to shield off the biological samples during the staining. In this known technique, one risk is the drying out of slides and lack of control of airspeed and temperature.
On this background, it is an object of the invention to provide an automatic pre-treatment of the biological samples on slides or other similar carriers or substrates, in the automatic staining apparatus so that the entire processing of the biological samples may be performed in a single automatic apparatus.
One of the various aspects that may be significant to users of automated process systems is that of allowing changes to the processing while it is ongoing. In this regard, it has often been considered that operators have to allow existing sequences to finish before inserting or changing the aggregate in some manner. In addition, operators often have needed particular knowledge and skills in order to assure the integrity of the process or instrument or result. The present invention seeks to reduce such effects to some degree and seeks to provide a system that may be considered more user, operator, supplier, or manufacturer friendly and may be adaptable to real-world conditions and events.